A growing wealth of data indicates that targeted therapies can mobilize a patient's own immune system to destroy malignancies with fewer side effects than traditional chemotherapy. Since it is estimated that 41% of Americans—almost 1 in 2 people—born in 2011 will develop cancer in their lifetime,8 the generation of more effective cancer immunotherapies is a high priority. These therapies include monoclonal antibodies (mAbs) that direct innate immune cells to tumor-associated antigens (TAA) as well as cancer “vaccines” that take many forms (including injections of tumor proteins with adjuvants or ex vivo primed dendritic cells) and are designed with the intention of inducing long-lasting anti-tumor T-cells.1 The objective of the present inventors' research is to develop novel compounds capable of stimulating both innate and adaptive immune responses against tumors, thereby combining the immunologic strengths of both mAbs and cancer vaccines in a small molecule format.
To date, mAbs have demonstrated the greatest clinical efficacy of all cancer immunotherapeutics, with nine unconjugated mAbs against TAA approved by the FDA over the past 15 years.9 To capitalize on this therapeutic success but reduce the disadvantages of mAbs (expensive to produce, can provoke allergic reactions, lack oral bioavailability), the present inventors have developed small-molecule antibody recruiting molecules (ARMs)2. ARMs take advantage of the high prevalence of preexisting antibodies against dinitrophenol (DNP) in human serum, possibly caused by pesticide exposure. These molecules redirect anti-DNP antibodies to prostate cancer and other cancer cells expressing prostate specific membrane antigen (PSMA). PSMA is a membrane protein expressed at high abundance in human prostate carcinoma, but present at low levels in normal prostate and non-prostatic tissues.10 We have shown that ARMs are able to mediate prostate cancer destruction both in vitro2 and in vivo11 through both antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) by innate effector cells.
While the traditional view holds that mAbs act primarily through the innate immune system, burgeoning evidence highlights their link to adaptive immunity.12 Antibody-opsonized tumors or soluble TAA immune complexes induce phagocytosis or endocytosis through Fcγ receptors (FcγRs) on dendritic cells (DCs).3, 4 This mode of internalization leads to increased presentation of TAA peptides on MHC I and II, increased costimulatory molecule expression, and increased generation of antigen-specific CD4+ and CD8+ T-cells as compared to unopsonized antigen.13-16 In mice, anti-CD20 and anti-Her2/neu mAbs were shown to induce adaptive anti-tumor immunity,17,18 while in humans, clinical response following anti-Her2/neu therapy correlates with greater increases in endogenous anti-Her2/neu antibodies and anti-tumor CD4+ T-cells.19,20 
Unfortunately, across all monoclonal antibodies used for tumor therapy, only about 30% of treated patients experience increased progression-free or overall survival.20,21 The tumor microenvironment can be highly immunosuppressive, characterized by release of anti-inflammatory paracrine factors, consumption of metabolites necessary for Teff proliferation, and recruitment of tolerogenic cells such as regulatory T-cells (Tregs).5 Many tumors are also poorly immunogenic in that, despite their malignant transformation, they consist of mostly self-proteins to which the immune system is tolerant Immunogenic epitopes arising from mutated gene products, expression of oncofetal proteins, or aberrant posttranslational modifications may represent only a small fraction of a tumor's total protein. Clearly, additional immuno stimulation beyond unmodified anti-tumor mAbs is required to mount a successful adaptive immune response in the majority of patients.
Advances in the cancer vaccine field have underlined the importance of adjuvants in generating immunity. Toll-like receptor (TLR) agonists are particularly powerful adjuvants, since the natural ligands of these receptors are highly conserved pathogen-associated molecular patterns that are recognized as primal danger signals by the immune system.22 pursuant to the present invention, the inventors propose to attach the synthetic small-molecule TLR7/8 agonist imiquimod and related TLR7/8 agonists to the ARM scaffold. Located in endosomes, TLRs 7/8 mediate production of proinflammatory cytokines and induce maturation of antigen presenting cells (APCs).6,7 TLR7/8 agonists can heighten innate responses, especially through plasmacytoid dendritic cells, as well as potentiate adaptive Th1 polarization and cytotoxic T-cell (CTL) generation.23 Imiquimod is already FDA-approved for topical use against skin malignancies and has also been tested systemically as an experimental vaccine adjuvant.24,25 It is the present inventors' view that the conjugation of imiquimod to an ARM will better stimulate APC activation and generate anti-tumor T-cells while also improving the ARM's ability to mediate tumor lysis by innate immune cells.
The direct conjugation of imiquimod and other TLR7/8 agonists to the ARM offers crucial advantages over co-administration of unlinked molecules. Several mouse studies have shown that antigen-specific CD4+ and CD8+ T-cell generation dramatically improved when injected antigen was directly linked to TLR7/8 agonists.26-28 Systemically administered TLR7/8 agonists in human trials have provoked significant adverse effects including fever, fatigue, nausea, chills, and myalgia without demonstrating much efficacy.29, 29, 30 Physical conjugation to the larger ARM should (1) increase the therapeutic index of the TLR7/8 agonist's (e.g. imiquimod's) therapeutic index by preventing its interaction with endosomal TLR7/8 until phagocytosis of an ARM-opsonized target cell has occurred and (2) localize the immunostimulant to the tumor site. Beyond this superior pharmacokinetic profile, there is some evidence that control of antigen presentation is phagosome-autonomous, meaning that TLR agonists must be located in the same endosome as phagocytosed TAA to best enhance TAA peptide presentation on MHC I or II.27, 31, 32 Extensive preclinical and clinical data thus support the conjugation of TLR7/8 agonists to antibody recruiting molecules for greater induction of anti-tumor immunity.